EXCEED THE SPACE PROVIDED. Our studies have sought to understand the function and regulation of ABC transporters with a focus on the MDR1 gene that encodes a protein conferring resistance to a large number of therapeutic drugs and cytotoxic compounds. During this grant cycle we identified a mitochondria! ABC transporter that was increased in rat liver tumors, initially referred to as PRP for P-glycoprotein related p/otein. In the current application we have extended these studies and isolated the human orthologue, referred to as ABCB6 and two new alternate forms we call HuPRP and HuPRP-2. We show that ABCB6 is overexpressed in human liver, lung and colon tumors and that it increases colony survival, strongly indicating ABCB6 has an antiapoptotic role. This is consistent with the phenotype of yeast that have either lost or have impaired function of the orthologous gene, ATM1. Loss of the gene product, Atmlp, results in a constellation of defects including: impaired growth, mitochondrial respiratory defects, increased iron accumulation and compensatory changes in glutathione, as well as a loss of holoenzymes and an accumulation of apoenzymes of heme-containing proteins. Thus, while Atmlp functions to maintain mitochondrial iron homeostasis its loss precipitates effects leading to impaired growth and survival. ABCB6, also has a known mitochondrial function, i.e.,maintenance of iron homeostasis, which is critical in cellular respiration. However, we demonstrate that HuPRP and HuPRP-2 overexpression, impairs growth and induces apoptosis while ABCB6 promotes cell survival. Moreover, since ABCB6 and HuPRP physically interact, it is conceivable that alterations in the balance between these forms lead to mitochondrial impairment and cell death. Further ABCB6, HuPRP, and HuPRP-2, are ubiquitously expressed, a finding that suggests a balance exists normally between these forms and this is linked to a genetic recessive disorder (lethal neonatal metabolic syndrome") that has both impaired mitochondrial function and iron accumulation in tissues such as liver and macrophage and is localized to the same chromosomal region as ABCB6. Because the cell death caused by HuPRP is substantially rescued by the anti-apoptotic gene, Bel-XL it implies that the Bcl-2 pathway is involved in the Hu-PRP forms of cell death. We will test the following: 1) determine the physiological role of ABCB6 in the intact animal by developing an ABCB6 knockout, 2) evaluate ABCB6 role in carcinogenesis using a transgenic mouse model, 3) determine the role of ABCB6 in increased survival, 4) role of the proapoptotic Bcl-2 family members, BAX/BAK in Hu-PRP induced cell death using selective knockout models. Cumulatively, these studies will lead to better understanding of this important mitochondrial ABC transporter in mitochondrial dysfunction and diseases such as cancer. ./"